10 research outputs found
Influence of l-carnosine on pro-antioxidant status in elite kayakers and canoeists
Carnosine is a dipeptide formed from the amino acids β-alanine and histidine and found in large amounts in the brain and muscle, especially fast twitch muscle. Carnosine has an antioxidant role and accounts for about 10% of the muscle’s ability to buffer the H+ ions produced by high intensity exercise. Due to the interesting role of carnosine, the aim of the study was observe the effects of carnosine intake on pro-antioxidant status in highly trained athletes exposed to intense exercise.Fourteen male athletes from the Polish national kayak and canoe teams participated in placebo-controlled and cross-over study. The athletes were supplemented with 4 g/d carnosine for 14 days. Blood samples were collected before and 30 min, 24 h and 48 h after 2000 m exercise trial. In blood, hydrogen peroxide (H2O2), nitric oxide (NO), markers of RO/NS activity 8-isoprostanes and 3-nitrotyrosine, total (GSHt) and oxidised glutathione (GSSG), antioxidant status (APO) and superoxide dismutase (SOD) were determined. There were not observed statistically significant differences in exercise-induced changes in H2O2 and NO concentrations and SOD activity after carnosine intake. However, carnosine prevented an increase in 8-isoprostanes, 3-nitrotyrosine and GSSG concentrations as well as elevated redox status (GSHt-2GSSG)/GSSG at post-exercise period.Although, oral supplementation with 4 g carnosine did not affect RO/NS generation, it significantly attenuated exercise-induced glutathione loss, reduced oxidation/nitration markers concentration and SOD activity. These results suggest that carnosine could provide antioxidative protection for highly trained athletes
The Molecular Engineering of an Anti-Idiotypic Antibody for Pharmacokinetic Analysis of a Fully Human Anti-Infective.
Anti-idiotype monoclonal antibodies represent a class of reagents that are potentially optimal for analyzing the pharmacokinetics of fully human, anti-infective antibodies that have been developed as therapeutic candidates. This is particularly important where direct pathogen binding assays are complicated by requirements for biosafety level III or IV for pathogen handling. In this study, we describe the development of a recombinant, anti-idiotype monoclonal antibody termed E1 for the detection of a fully human, serotype-specific, therapeutic antibody candidate for the BSLIII pathogen Dengue virus termed 14c10 hG1. E1 was generated by naïve human Fab phage library panning technology and subsequently engineered as a monoclonal antibody. We show that E1 is highly specific for the fully-folded form of 14c10 hG1 and can be employed for the detection of this antibody in healthy human subjects' serum by enzyme linked immunosorbent assay. In addition, we show that E1 is capable of blocking the binding of 14c10 hG1 to dengue virus serotype 1. Finally, we show that E1 can detect 14c10 hG1 in mouse serum after the administration of the therapeutic antibody in vivo. E1 represents an important new form of ancillary reagent that can be utilized in the clinical development of a therapeutic human antibody candidate
Expression and characterisation of E1.
<p>(A) Schematics showing the difference in the hinge structure between mG2a and mG2a/hG3. (B) 12% SDS-PAGE gel (reducing and non- reducing) showing the expression of E1 mG2a/hG3 and E1 mG2a (left) and E1 Fab (right). The heavy chain and light chain bands correspond to sizes, 50kDa and 25kDa respectively in the reducing gel. (C) Direct ELISA comparing the binding profile of E1 mG2a and E1 mG2a/hG3 for 14c10 hG1. The wells were coated with 14c10 hG1 and E1 mG2a and E1 mG2a/hG3 were added. Results represent average of two independent experiments performed in duplicates. Error bars represent standard error mean (SEM). (D) Determination of the binding specificity of E1 mG2a/hG3 for 14c10 hG1. The antigens (14c10 hG1, 3H5hG1, D29 hG1 and commercially available human IgG1) were coated and the detector E1 mG2a/hG3 was used, (E) the antigens (14c10/3H5/D29 hG1) with and without purified human polyclonal IgGs were added to the coated E1 mG2a/hG3. Results are representative of two independent experiments performed in duplicates. Error bars represent SEM. (F) Inhibition of binding of 14c10 hG1 to DENV-1 with E1 Fab. The wells were coated at a constant concentration with 4G2 mG2a as a capture for DENV-1. The antibodies (14c10 hG1 and D29 hG1) were used at a constant concentration and were preincubated with E1 Fab at room temperature, before the addition to DENV-1. Results are an average of two independent experiments performed in duplicates. Error bars represent SEM.</p
Determination of the detection limit of E1 mG2a/hG3 for 14c10 hG1 in human serum.
<p>In the sandwich ELISA assay format, the wells were coated with E1 mG2a/hG3 as a capture. Ten different healthy subjects’ sera were used at different dilutions and spiked with 14c10 hG1. Each graph is representative of the average of at least two independent experiments. Error bars represent the SEM.</p
Discovery of anti-idiotypic antibody against 14c10 hG1.
<p>(A) Schematics of phage library panning which consists of four main steps; the binding of Fab expressing phage to the coated target antigen (14c10 Fab), the washing away of the unbound phage, followed by the elution and amplification of the bound phage. The cycle then repeats for the enrichment of the specific phage for the target antigen. (B) Polyclonal ELISA showing the enrichment of the successive rounds of phage panning against 14c10 Fab. The wells were coated with 14c10 Fab, other irrelevant Fabs (3H5 and D29) and blocking buffer (skim milk). Negative control was the addition of skim milk instead of the polyclonal phage to the coated antigens. Results represent one independent experiment. (C) Monoclonal ELISA showing the specificity of the monoclonal phage from pan three. Controls were included to ensure that the antigens were properly coated; negative control and positive control were the addition of skim milk instead of the monoclonal phage clones and detected with αM13- HRP (negative) and α-histag-HRP (positive) respectively. Results represent one independent experiment. (D) ELISA to test the specificity of phage clone E1 for 14c10 hG1 in serum. The antigens (14c10 hG1, 3H5 hG1 and D29 hG1) were coated. PEG precipitated phage clone E1 was used at 1 in 10 dilution either in human serum (diluted 1 in 4 with skim milk) or skim milk. Results represent one independent experiment.</p